Functions And Toxins Delivery Mechanisms Of PAAR Proteins In Myxococcus Xanthus

Microbe inhabit complex environments,where they interact and compete with other organisms.Multitudinous offense and defense systems emerged during evolution to combat resource competitors and parasites.These systems are involved in incessant arms races to fight for territory and nutrition.Microbe usually deploy protein toxins in conflicts with other organisms.Many protein toxins are also a critical factor in the pathogenicity of many bacterial pathogens that threaten the health of humans,animals or plants.Therefore,the protein toxins secreted by microorganisms have important value on natural ecosystems and human health.Type VI secretion system（T6SS）is an important apparatus for secreting protein toxins widely distributed in Gram-negative bacteria.The T6SS toxin effector participates in many important physiological processes,for example,interbacterial warfare,and pathogenesis toward humans,animals and plants.T6SS is a complex nanomolecular machine composed of at least 13 core components.The T6SS shares structural homology to the T4 contractile bacteriophage tail,in which Hcp（hemolysin co-regulated protein）inner tube,VgrG（valine-glycine repeat protein G）and PAAR（proline-alanine-alanine-arginine）protein form a puncture complex used to deliver toxin effectors,and the toxin is delivered into adjacent cells by injections.The PAAR protein is conical in structure and is located at the T6SS tip to sharpen the injector for piercing the target cell envelope.Inactivation of paar genes will not interfere with T6SS functions,but may result in the inability to kill specific target cells.Therefore,the PAAR protein is considered to be an important effector carrier.Few PAAR-related toxins have been identified,but they have shown complex secretion patterns,such as direct fusion as the C-terminal extension domain of the PAAR protein,or non-covalent binding with the PAAR protein to form a complex.It is of great significance to study the PAAR protein and mechanisms of PAAR proteins delivering toxins.Myxococcus xanthus DK1622 is a model strain of myxobacteria and the genome has been sequenced completely.We previously performed random insertion mutations in the genome of DK1622 and obtained 9 independent mutants.These mutants not only formed a visible colony boundary with DK1622,but also formed a clear colony boundary between the mutant strains.However,colony merger could occur between their own strains.The insertion sites were scattered in the DK1622 genome.The insertion genes from five colony-merger deficient mutants were homologous,and the genes immediately upstream of these five genes were also homologous.Experiments have shown that the inserted gene homologues encoded a class of immunity proteins.The upstream gene homologues encoded a class of AHH nuclease toxin effectors.The immunity gene deletion mutants formed colony boundaries with the wild type strain DK1622,and the survival of deletion mutant was greatly impacted by the wild type strain,and the deletion mutant and DK1622 strain could not co-exist.The DK1622 genome encodes a functional T6SS gene cluster（MXAN₄800-MXAN₄813）,and with the correlated method of cryogenic photoactivated localization microscopy and cryo-electron tomography,the T6SS structure has been identified and visualized in M.xanthus cells.We identified six paar genes in M.xanthus genome far away from the T6SS gene cluster.There were some homologous genes with unknown functions in the upstream and downstream of the six paar genes,constituting six paar gene clusters.The six PAAR proteins were divided into two groups:four were single-domain PAAR proteins,while the other two were multidomain PAAR proteins containing the N-and C-terminal domains.The four single-domain paar gene clusters are related to the insertion site genes of mutants that formed colony boundaries with wild strains.There were the nuclease toxin-immunity gene pairs upstream or downstream of the PAAR encoding genes.A nuclease toxin-immunity gene pair in one of the gene clusters had been experimentally proven,and both PAAR and T6SS were necessary for the delivery of the nuclease toxin.It was unclear whether the gene neighbors of the other two multidomain paar gene encode toxin effector-immunity protein system,and it was also unclear how the six paar genes,which are far from T6SS gene cluster,contribute to the secretion of the toxin effectors.We further explored the functions of multidomain PAAR proteins and the toxins delivery mechanisms of single-domain PAAR proteins.The PAAR domains were located at the N-terminus of the multidomain PAAR proteins in DK1622,and the sequences were highly similar.However,there were no sequence homology and known domains detected in the C-terminal extended domains（CTD）.In addition to the similar N-terminal PAAR domain,the gene neighbors of these two PAAR-CTD proteins in the genome were also highly similar.Two paar-ctd genes located in two homologous operons involved in different ecological functions:one has antifungal activity,and the other is associated with the self-identification phenotype.These two PAAR-CTD proteins were both toxic,and their downstream adjacent genes protected against the toxicities.The PAAR-CTD proteins and the proteins encoded by their downstream genes form two toxin-immunity systems.We further identified many paar-ctd gene loci with similar sequences in other myxobacteria genomes,and the PAAR proteins had at least 7 different C-terminal domains.Therefore,the results indicated that the paar gene loci with highly similar sequences can achieve great functional changes by different C-terminal sequences.Related toxins of multidomain PAAR proteins were delivered as C-terminal domains.We further analyzed the toxins delivery mechanism of the four single-domain PAAR proteins in M.xanthus DK1622.The four paar genes also had similar gene neighbors in the genome,and there were the nuclease toxin-immunity gene pairs around them.These four gene clusters might have similar working modes.We focused on PAAR protein MXAN₀044 and its gene neighbors.We found that MXAN 0044 gene and MXAN₀045 gene function in the colony-merger incompatibility in M.xanthus cells,which might be associated to the delivery of MXAN 0050 nuclease.There was no direct binding ability between the MXAN 0044 and MXAN 0050 proteins,but there was the binding ability between the MXAN 0044 and MXAN₀045 proteins,also between the MXAN₀045 and MXAN₀050 proteins.The MXAN₀045 protein formed a complex with the toxin and at the same time weakens the nuclease activity of toxin,thereby indirectly protecting immunity protein-deficient cells.Therefore,the results indicated that the toxins delivery mechanism of the single-domain PAAR protein is complicated,and MXAN-0045 may be a new class of adaptor proteins that can mediate the binding of PAAR proteins to toxins.We found that the single-domain PAAR protein of M.xanthus required the new adaptor MXAN₀045 protein to deliver toxins.Adaptor proteins were mediators that help to load their cognate effectors onto the T6SS spike complex（PAAR or VgrG protein）.We identified the three classes of known adaptors（DUF1795,DUF2169 and DUF4123）and a new class of adaptors on a large scale in all reference or representative prokaryotic genomes.The contextual genes of the three classes of known adaptor proteins all exhibited a high proportion of encoding T6SS spike complex protein and effector proteins.According to the characteristic of genomic context,we further identified a new class of adaptor protein（PRK06147）.And the MXAN₀045 protein we identified in M.xanthus DK1622 belongs to this protein family.The four classes of adaptors had no homology in sequence,and were clearly different in protein size.They are widely distributed among the bacterial genomes.From neighbors of 5297 adaptor genes,we identified 1356 potential effector genes from 92 different families,and two-thirds were currently annotated as hypothetical proteins or as having unknown functions.The majority of these annotated toxins were predicted to have enzymatic activity,and most of these acted on nucleic acids.The four classes of adaptors had different preferences for toxins.These results indicated that each class of adaptors can be used as an effective marker to identify T6SS toxic effectors.The six PAAR proteins encoded by M.xanthus DK1622 genome exhibited different functions and toxin delivery mechanisms.We further systematically analyzed PAAR proteins in prokaryotic genomes.More than 40,000 PAAR homologues were identified.The PAAR homologues could be divided into 8 different types and subdivided into 16 subtypes through phylogenetic relationships.There was no clear boundary between the PAAR proteins associated with T6SSs and extracellular contractile injection systems（eCISs）in the phylogenetic relationship.PAAR proteins from one subtype were versatile between the T6SSs and eCISs.We also detected more than 80 families of toxins associated with PAAR proteins.One PAAR subtype can be associated with toxins of over 40 families,and toxins from one family can be associated with more than 10 PAAR subtypes.A large-scale comparison of Earth Microbiome Project（EMP）data and prokaryotic genomes information revealed that the prokaryotes encoding paar genes were widely present in diverse environments worldwide,and the taxa encoding multiple copies of the paar gene exhibited higher abundance and wider distribution in environments than other taxa.The results highlighted that PAAR proteins greatly enrich the weapon arsenal of prokaryotes and help prokaryotes fight for survival advantages in a crowded environment.Our comprehensive and systematic analysis of the PAAR protein and the toxins delivery mechanisms of the PAAR proteins revealed the important role of PAAR protein in the struggle for existence of prokaryotes.The M.xanthus multidomain PAAR proteins with different C-terminal sequences greatly enriches the arsenal of M.xanthus and expands their ability to attack surrounding neighbors.PAAR proteins were versatile between the T6SS and eCIS,and there was a great diversity of toxins associated with PAAR proteins.We discovered a new class of adaptor proteins by studying the toxins delivery mechanism of single-domain PAAR protein,and systematically analyzed four classes of adaptor homologues,enriching our understanding of the PAAR-dependent toxin delivery mechanisms.Based on the analysis of the PAAR protein,thousands of toxin genes have been identified,most of which have unknown functions.These new discoveries contribute to increase the understanding of the prokaryotic toxin systems.These results might have important implications for the assembly of protein machinery,pathogenicity of pathogenic bacteria,and the role of microbial communities.The new toxins and immunity proteins identified may also have potential applications in biotechnology.